Embryonic stem cells are pluripotent stem cells derived from the inner cell mass of embryo in blastocyst phase. They can be induced to differentiate into all three germ layer cells and have great potential for clinical applications including drug development, cell sources for tissue engineering and cell therapy. However most of human embryonic stem (HES) cells are cultured in the presence of either mouse embryonic fibroblasts (MEF) or human cells nowadays. Thomson et al. (U.S. Pat. No. 5,843,780) were the first to successfully culture stem cells from primates and human. They cultured HES cells on a layer of feeder cells, which were mouse embryonic fibroblasts that serve as an adherent layer for the stem cells and as a source of nutrients, bioactive material, and growth factors.
Nowadays, most of HES cells are maintained in culture in an undifferentiated state on a layer of inactivated feeder cells originated from either mouse or human. Some problems exist in a feeder layer dependent culture system for manipulation and clinical application of HES cells, for example, (1) the potential risks of transmitting pathogens from the animal feeder cells to the HES cells during cell therapy, (2) feeder cells are mainly from primary cells, while primary cells from different batches offer different effect as feeder cells, rendering the quality control of the cultured HES cells more difficult; (3) the limited sources and numbers of feeder cells hamper the mass production and applications of HES cells. Therefore, it is desirable to maintain the undifferentiated growth of HES cells without using the feeder cells for mass production and clinical application of HES cells.
Xu et al (Nat. Biotechnol., 19(10):971-974, 2001; WO 03/020920 and U.S. 2003/0017589) were the first to successfully maintain undifferentiated growth of HES cells in a feeder-free culture system. In this system, HES cells are cultured on Matrigel® or laminin in a medium pre-conditioned by mouse embryonic fibroblasts (MEF). Matrigel is a solubulized basement membrane preparation extracted from mouse sarcoma, a tumor rich in extracellular matrix (ECM) proteins. It has the potential risk of transmitting xeno agents during clinical applications of HES cells. Furthermore, such matrices and defined-matrix macro-molecules as of Matrigel are insufficient in mimicking the more complex cell-martix interactions provided by the feeder cells. Another study has also indicated that this culture system is only suitable for certain HES cell lines, e.g. H1 and H9, but unsuitable for other HES cell lines (Richards, M. et al., Nat Biotechnol., 20(9):933-936, 2002).
Accordingly, it is an object of the present invention to provide an alternative feeder-free culture system to overcome the afro-mentioned problems encountered in the prior art such as potential pathogen transmitting during cell therapy, poor quality control and etc., and to grow substantially undifferentiated HES cells for periods sufficient to allow the production of HES cells for applications such as cell therapy, drug discovery and gene therapy.